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A kind of high-yielding farnesene recombinant bacteria and its construction method and application

A technology of farnesene and recombinant bacteria, applied in the field of microorganisms, can solve problems affecting product yield, affecting bacterial cell stability expression and activity production, etc., achieving high yield and purity, reducing the risk of bacterial contamination, and improving yield

Active Publication Date: 2022-08-09
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, medium composition (carbon-to-nitrogen ratio), stirring speed, dissolved oxygen, temperature, pH, feeding composition and method, etc. will all affect the growth of bacteria, the stability of plasmids, the expression and activity of enzymes, the generation of by-products, etc. , thus affecting the yield of the product

Method used

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  • A kind of high-yielding farnesene recombinant bacteria and its construction method and application
  • A kind of high-yielding farnesene recombinant bacteria and its construction method and application
  • A kind of high-yielding farnesene recombinant bacteria and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Construction method of recombinant bacteria with high production of farnesene.

[0042] 1) Construction of plasmid pTrcLower-ΔIDI: Using the existing plasmid pTrcLower in the laboratory as a template, the part of the vector except the ScIDI gene was amplified by PCR. The upstream and downstream primers GA-Low-F and GA-Low-R sequences are as SEQ ID No. :15-16, the PCR system is as follows:

[0043]

[0044] The PCR product was subjected to agarose gel electrophoresis and the target band was recovered by gel tapping. The target fragment pTrc-ERG12-ERG8-ERG19 was about 8390bp. The recovered product undergoes self-ligation reaction:

[0045]

[0046] The ligation product was mixed with 5 μL of sterile water, and all DH5α competent cells were heat-shocked and coated on LB Amp plates, and cultured at 37°C overnight. The next day, the colonies on the plate were observed, and a single colony was picked into the liquid medium, cultivated at 37°C to a relativel...

Embodiment 2

[0062] Example 2. Application of the recombinant bacteria constructed in Example 1 in the synthesis of farnesene.

[0063] The quantitative detection of farnesene by gas chromatography described in this example, the chromatographic column is an Agilent DB-5MS (30m × 0.25mm × 0.25μm) capillary column, and the column heating program is: initially at 60°C for 0.75min, and at 40°C for 0.75min. The temperature was raised to 300°C for 2 min at a rate of °C / min, and then decreased to the initial temperature. Use β-farnesene standard as a standard curve (y=3056.9x, x is the concentration of β-farnesene standard, the unit is g / L; y is the peak area of ​​β-farnesene) for quantification.

[0064] The primary seed medium described in this example is LB medium, and its components are: 10 g / L NaCl, 10 g / L peptone, 5 g / L yeast extract, and the rest is water.

[0065] The composition of the secondary seed medium (fermentation medium) is: 20g / L glucose, 9.8g / L K 2 HPO 4 , 5g / L beef extract,...

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Abstract

A high-yielding farnesene recombinant bacterium and a construction method and application thereof belong to the technical field of microorganisms. In order to further improve the yield of Escherichia coli synthesizing farnesene, and improve the catalytic efficiency and stability of enzymes on the synthetic pathway, the present invention provides a genetically engineered bacterium for synthesizing farnesene, wherein the genetically engineered bacterium overexpresses mvaE, mvaS, ERG12, ERG8, ERG19, AaIDI, ispA, and β-farnesene synthase D18-AaFS gene with D18-localized peptide. The farnesene yield of the genetically engineered bacteria reaches 35.93 g / L in 96 hours of fermentation, which is beneficial to promoting the industrialization process of biosynthesis of farnesene.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a recombinant bacterium with high production of farnesene and a construction method and application thereof. Background technique [0002] Farnesene, molecular formula C 15 H 24 , also known as farnesene, is a chain sesquiterpene. Farnesene can be used as an additive in daily chemical, pharmaceutical, food and other industries due to its aromatic odor and antioxidant activity; it can also be used as a pheromone for biological control of agricultural pests; in addition, farnesene is also a synthetic vitamin E An important intermediate in the side chain. In recent years, terpene-based biofuels have received increasing attention. The hydrogenation product of farnesene, farnesane, is considered to be a promising new biofuel in the aerospace field due to its high cetane number and low carbon emissions, and has passed the test of ASTMD7566 aviation fuel standard ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/53C12N15/54C12N15/60C12N15/61C12P5/02C12R1/19
CPCC12N15/52C12N9/1029C12N9/0006C12N9/1025C12N9/1205C12N9/1229C12N9/88C12N9/90C12N9/1085C12N15/70C12P5/026C12Y203/01016C12Y101/01088C12Y203/0301C12Y207/01036C12Y207/04002C12Y401/01033C12Y503/03002C12Y205/01092C12Y402/03047C12N2800/22Y02E50/10
Inventor 张海波门潇咸漠刘晋锋
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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